Combinatorial approach to develop novel pre-therapeutic agents targeting virulence factors essential to clinically relevant pathogens

开发针对临床相关病原体必需毒力因子的新型治疗前药物的组合方法

• 批准号: 10681469
• 负责人: Hanh Ngoc Lam
• 金额: $ 24.07万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-09 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10681469
• 关键词: Active Sites; Affinity; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Aspartate; Bacteria; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biology; C-terminal; Calorimetry; Cause of Death; Cell Death; Cell membrane; Cells; Cessation of life; Chemicals; Collaborations; Communicable Diseases; Computer Assisted; Consumption; Data; Development; Drug resistance; ESKAPE pathogens; Enzyme Activation; Fluorescent Probes; Foundations; Future; Goals; Gram-Negative Bacteria; Health; Health Care Costs; Host Defense Mechanism; Hybrids; Infection; Inflammation; Knowledge; Learning; Legal patent; Libraries; Medicine; Membrane; Mentors; Methods; Microbe; Molecular Target; Mus; N-substituted Glycines; Natural Products; Nosocomial Infections; Outcome; Pathogenesis; Pathogenicity; Pathology; Peptides; Peptoids; Periodicity; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Phenotype; Phospholipase A2; Process; Productivity; Proteins; Pseudomembranous Colitis; Pseudomonas; Pseudomonas aeruginosa; Regulation; Research; Resistance; Resolution; Series; Serine; Serine Hydrolase; Specificity; Structure; Structure-Activity Relationship; Techniques; Therapeutic; Time; Titrations; Type III Secretion System Pathway; United States; Virulence; Virulence Factors; Virulent; Work; X-Ray Crystallography; acute infection; algorithm development; antibiotic resistant infections; antimicrobial; antimicrobial drug; appendage; clinically relevant; cofactor; combat; combinatorial; commensal microbes; cost; cost effective; design; drug candidate; drug discovery; extracellular; genetic approach; global health; healthcare-associated infections; high throughput screening; inhibitor; innovation; insight; lead optimization; macromolecule; marine; microbial; microbiome; microbiota; multidrug-resistant Pseudomonas aeruginosa; mutant; novel; novel strategies; novel therapeutics; pathogen; pathogenic bacteria; pressure; prevent; priority pathogen; screening; skills; small molecule; targeted treatment; therapeutic target; trait; virtual screening

Project Summary/Abstract Antibiotic resistance is a serious global health threat. Current antibiotics target essential bacterial processes and impose strong selective pressure for resistance. Upon antibiotic treatment, the healthy microbiota are reduced in both number and diversity, leading to serious health consequences such as Clostridium difficile colitis. Moreover, resistant traits can be transferred to other microbes, leading to the spread of antibiotic resistance. Using virulence blockers to target specific pathogenicity mechanisms, while leaving the microbiota intact, is a promising strategy to reduce resistance. This proposal will identify molecular target of a newly discovered the type III secretion system (T3SS) inhibitor, and explore their modes of action for further optimization and development. I will assess structure – activity relationship to optimize the T3SS inhibitors, cyclic pepeptomers, and use affinity based method to identify their molecular targets in Pseudomonas aeruginosa. Besides, target-based drug discovery offers the advantage of being low cost and less time consuming. With the availability of high-resolution structure and development of algorithms to predict binding affinity and poses of small molecules to its protein target, virtual screening can provide lead for optimization. ExoU, an effector with phospholipase A2 activity, is the major effector in P. aeruginosa, one of six ESKAPE pathogens which cause the majority of nosocomial infections in the U.S. and “escape” antimicrobial drugs. ExoU has a serine/aspartate catalytic dyad and a separate cofactor-binding domain required for activation of the enzyme. ExoU is highly toxic, associated with acute infection, antibiotic resistance and severe outcome in patents. Delay ExoU expression can increase mice survival. Thus we set out to find ExoU inhibitors as a strategy to treat acute infection and reduce resistance. We will identify ExoU inhibitors that 1) inhibit enzymatic activity by targeting its catalytic residue serine, or 2) bind to the membrane localization domain which will prevent ExoU's activation by virtual screening. The inhibitors that show binding affinity to ExoU in isothermal titration calorimetry assay, and prevent cell death caused by ExoU will be chosen for optimization. Structure of inhibitor-bound proteins will be solved using X-ray crystallography. I believe that my team of mentors (Drs. Stone and Ottemann), advisors (Dr. Rubin, expert in X-ray crystallography; Dr. Jacobson, expert in computer- aided drug discovery) and collaborators (Dr. Lokey, an expert in macromolecule synthesis, Drs. Crews and Linington, natural product chemists) will provide me support to successfully carry out this project. With the biochemical techniques I will learn, the structures and new inhibitors I will obtain in the K99 phase, I will then collaborate with Dr. Shaw (medicinal chemist) and Dr. Jacobson to optimize the candidate hits in my independent phase. This project extends my skill set in biochemical methods and has the potential to provide substantial momentum towards drug discovery, and development. These will serve as the foundation of a R01 proposal to be prepared upon the completion of the main stages of this research plan.

项目概要/摘要 抗生素耐药性是一个严重的全球健康威胁。目前的抗生素针对重要的细菌过程 并对抵抗施加强大的选择压力。经过抗生素治疗后，健康的微生物群 数量和多样性均减少，导致严重的健康后果，例如艰难梭菌 结肠炎。此外，耐药性特征可以转移到其他微生物，导致抗生素的传播 反抗。使用毒力阻断剂针对特定的致病机制，同时保留微生物群 完好无损，是减少阻力的一个有前途的策略。该提案将确定新的分子靶标 发现了III型分泌系统（T3SS）抑制剂，并进一步探索其作用方式 优化和发展。我将评估结构-活性关系以优化 T3SS 抑制剂， 环状肽异构体，并使用基于亲和力的方法来鉴定其在假单胞菌中的分子靶标 铜绿假单胞菌。此外，基于靶标的药物发现具有成本低、时间短的优势 消耗。随着高分辨率结构的可用性和预测结合算法的开发 小分子与其蛋白质靶点的亲和力和姿势，虚拟筛选可以为优化提供线索。 ExoU 是一种具有磷脂酶 A2 活性的效应子，是铜绿假单胞菌（六种 ESKAPE 之一）的主要效应子 引起美国大多数医院感染的病原体并“逃避”抗菌药物。 ExoU 具有丝氨酸/天冬氨酸催化二元体和激活 酶。 ExoU 具有剧毒，与急性感染、抗生素耐药性和严重后果有关 专利。延迟 ExoU 表达可以增加小鼠的存活率。因此我们开始寻找 ExoU 抑制剂作为 治疗急性感染和降低抵抗力的策略。我们将鉴定 ExoU 抑制剂，1) 抑制酶 通过靶向其催化残基丝氨酸来提高活性，或 2) 与膜定位结构域结合，这将 通过虚拟筛选阻止ExoU的激活。在等温条件下显示出与 ExoU 结合亲和力的抑制剂 选择滴定量热法进行优化，防止ExoU引起的细胞死亡。结构 抑制剂结合蛋白将使用 X 射线晶体学来解析。我相信我的导师团队（Drs. Stone 和 Ottemann），顾问（Rubin 博士，X 射线晶体学专家；Jacobson 博士，计算机专家） 辅助药物发现）和合作者（大分子合成专家 Lokey 博士、Crews 博士和 Linington，天然产物化学家）将为我成功开展这个项目提供支持。随着 我将学习的生化技术，我将在K99阶段获得的结构和新的抑制剂，然后我将 与 Shaw 博士（药物化学家）和 Jacobson 博士合作，优化我的候选命中 独立相。该项目扩展了我在生化方法方面的技能，并有可能提供 药物发现和开发的巨大动力。这些将作为 R01 的基础 提案将在本研究计划的主要阶段完成后准备。